Novel nucleophile derivatives of cephalosporin c and allied compounds and their process of manufacture



United States Patent 3,278,531 NOVEL NUCLEOPHILE DERIVATIVES 0F CEPH- ALOSPORIN C AND ALLIED COMPOUNDS AND THEIR PROCESS OF MANUFACTURE James StuartGordon Cox, Radcliife-on-Trent, and Harry Fazakerley and John Derek Cooker, Chalfont St. Peter, England, assignors to Glaxo Laboratories Limited, Greenford, England, a British company No Drawing. Filed May 7, 1962, Ser. No. 193,015 Claims priority, application Great Britain, May 16, 1961, 17,845/ 61; Jan. 26, 1962, 3,026/ 62 18 Claims. '(Cl. 260-243) This invention relates to the production of novel der-ivatives of cephalosporin C and allied compounds.

The constitution of cephalosporin C has been interpreted in terms of the following structure:

0 (50 K I where R=(CH .CH'(NH )COOH and consequently the structure of the novel derivatives described below is presented in an analogous fashion.

It has now been found that cephalosporin C and allied compounds will react with certain compounds of a highly nucleophilic nature as hereinafter defined by displacement of the acyloxy group from the cephalosporin molecule to form novel derivatives thereof. Many of these derivatives have antibacterial activity which, in'some instances, is superior to that of the parent cephalosporin.

The compounds according to the invention have in general the important advantage of improved stability to degradiation in vivo (as evidenced, for example, by animal tests) compared with the corresponding acetoxy compounds. Since the latter possess activity against penicillin resistant organisms that is highly important. The compounds according to the invention are also of interest as intermediates for conversion to other cephalosporin derivatives.

According to the invention, therefore, there is provided a process for the production of derivatives of cephalosporin C which comprises reacting in a polar medium a compound of the general formula:

(in which R is an acyl group, particularly a lower alkanoyl group, and R and R are each hydrogen atoms or R is a hydrogen atom and R is an acyl group or a triaryl substituted alkyl group, e.g., a triphenyl methyl group or R and R together form a divalent acyl group derived from a dicarboxylic acid) or a salt thereof, With a strong nucleophile selected from:

(a) Thiourea and substituted thioureas including aliphatic, aromatic, alicyclic and heterocyclic substituted thioureas;

(b) Aromatic and aliphatic thioamides, e.g., thioacetamide and thiosemicarbazide;

(c) Thiophenol and substituted thiophenols;

(d) Substituted and unsubstituted primary and secondary aromatic amines, preferably free from tertiary nitrogen heterocyclic substituents;

(e) Thiols and substituted thiols, particularly amino thiols and substituted aminothiols;

"ice

(f) Metal salts, particularly alkali metal salts, of azide ion (-N hydrogen phosphate ion (HPO4=) and thio- HOOC.CH(NH (OH .CO

and the group R may be an acetyl group it will be appreciated that general Formula II includes cephalosporin C as Well as derivatives thereof.

Whilst R may represent an acyl group in general terms one may use other specific acyl derivatives representative of alkanoyl, alkenoyl, substituted alkanoyl, e.g., ,aralkanoyl, aryloxyalkanoyl, S-arylth-ioalkanoyl and S-aralkylthioalkanoyl, of cephalosporin derivatives of nucleophiles as defined herein. These acyl derivatives may be defined as having the general formulae:

(i) -R'(CH CO- Where R is aryl, cycloalkyl, substituted aryl or substituted cycloalkyl and n is an integer from l-4. Examples of this group include phenylacetyl, nitrophenylacetyl and phenylpropionyl.

(ii) C H CO where n is an integer from 2-7. The alkyl group may be straight or branched and, if desired, may be interrupted by an oxygen or a sulphur atom. Examples of such groups include hexanoyl, heptanoyl, octanoyl and butylthioacetyl.

(iii) C H CO Where n is an integer from 2-7. The alkenyl group may be straight or branched and, if desired may be interrupted by an oxygen or a sulphur atom. Examples of such groups include acrylyl, crotonyl and allythioacetyl.

(iv) R'OCR R"'.CO Where R has the meaning defined under (i) and R and R are the same or are different and each is a hydrogen atom or an alkyl group. An example of such a group is phenoxyacetyl.

(v) 'RSCR"R"'.C O where R, R" and R' are as defined above. Examples of such thio groups include S- phenylthioacetyl, S-chlorophenylthioacetyl and S-bromophenylthioacetyl.

(vi) R(CH S(CI-I ),,CR"R'.CO- where R, R" and R' are as defined above, m is an integer from l-4 and n is 0 or an integer from 14. Example of such a group include S-benzylt hioacetyl, benzylthiopropionyl and fi-phenethylthioacetyh (vii) R'CO- where R has the meaning defined above. Examples of such groups include benzoyl, substituted benzoyl and cyclopentanoyl. Where the benzoyl group is substituted the substituents may be alkyl or alkoxy and the substituents may be in the 2- or 2- and 6-posit-ion-s.

1A suitable 2, 6-disubstituted benzoyl group is therefore 2,6-dimethoxy-benzoyl.

The reaction may conveniently be effected by incubating the reactants in solution; that is, maintaining the reactants in solution at a moderate temperature, such as,

for example, l57() 0, preferably 37'50 C., for a pe-' riod of some hours or even days until the desired derivatained within the limits 5.0-8, preferably 67. If necessary the pH of the solution should be adjusted to the desired value by the addition of a bufiering agent such as sodium acetate or, when employing an alkali metal salt of the cephalosporin of general Formula II, by the addition of, for example, acetic acid.

Since the reaction appears to proceed by a polar or ionic mechanism it is necessary to employ a strongly polar medium for the reaction to proceed at a measurable rate. The most generally suitable solvent is water but in those cases in which the nucleophile is not very soluble in water a mixture of water and a water miscible organic solvent such as dimethylformamide, acetone or ethanol may be employed; suitable proportions for such solvent mixtures are 50:50 (v./v.)-or 30:70 (v./v.). Nonaqueous polar solvents such as acetone may also be used.

The reaction product may be separated from the reaction mixture, which may contain, for example, unchanged cephalosporin and other substances, by a variety of processes including crystallisation, ionophoresis, paper chromatography or by chromatography on ion-exchange resins.

If desired in some cases the compound obtained may then be incubated with a further nucleophile to effect displacement of the first nucleophile. This may be advantageous with certain nucleophiles.

Preferred cephalosporin compounds of general Formula II include cephalosporin C and its benzyl analogue (R =phenylacetyl), especially in the form of their alkali metal salts, for example the sodium salt. However as will be apparent from the examples below other such analogues have given rise to compounds having advantageous activity.

Suitable nucleophiles of group (a), the thioureas, include members of the general formula (in which R, R R and R may be same or diiferent and each represents a hydrogen atom or an alkyl, cycloalkyl, alkenyl, aryl, aralkyl group, or a substituted group of any of these types, or R and R together represent an alkylene group, e.g., an ethylene group). Examples of such thioureas includes thiourea itself, N-phenylthiourea, N,N'-diphenylthiourea and ethylene thiourea.

The substituted and unsubstituted primary and secondary amines (d) may be exemplified by aniline, p-nitro aniline, p-nitro-N-methyl aniline, sulphanilic acid and pamino benzoic acid; the group also covers the naphthylamines such as a-naphthylamine and substituted naphthylamines.

It is particularly preferred to use substituted thiols including aminothiols and substituted aminothiols as nucleophiles of group (e). Examples of such nucleophiles include 2-aminoethanethiol, 2-amino-2-methyl propane-1- thiol, 3-dimethylaminopropane-l-thiol and Z-piperidinoethane-l-thiol.

The anions employed as nucleophile in the form of their metal salts are preferably in the form of their alkali metal salts, e.g., sodium salts.

As previously mentioned the derivatives produced by the process according to the invention are new. The new compounds may be divided into the following general classes corresponding to the classes of nucleophiles set out above:

(21) Those having the general formula- (in which R R R R R and R have the meanings defined above).

(b) Those having the general formula- COzH (in which R and R have the meanings defined above and R is an aromatic or substituted aromatic radical) and their alkali metal salts.

This sub-class includes compounds derived from thiophenols substituted in the nucleus with an amino or substituted amino group, e.g., alkylamino or dialkylamino. Simple examples of thus thiophenols are therefore 0- and p-amino-thiophenol. It also includes compounds derived from thiophenols containing a conjugated electron attracting group, i.e., such a group in an 0- or p-position or, if desired, in more than one such position. Important examples of this type of nucleophile are various nitrothiophenols, e.g., o-nitrothiophenol and o,p-dinitro-thiophenol.

The term electron attracting group is, of course, wellknown in organic chemistry and refers to a substituent which attracts electrons more than hydrogen does (see for example A. M. Remick Electronic Interpretations of Organic Chemistry, John Wiley & Sons Inc., New York, 1943).

Examples of electron attracting groups which may be present on the nucleophiles used in the process according to the invention are nitro groups, nitroso groups, carbonyl groups, carboxyl groups, cyano groups and trifiuoromethyl groups.

((1) Those having the general formula (in which R R and R have the meanings defined above and R represent a hydrogen atom or an alkyl group) and their alkali metal salts;

(e) (i) Those having the general formula (in which R and R have the meanings defined above and R represents an aliphatic or, preferably, substituted aliphatic group) and their alkali metal salts;

(ii) Those having the general formula (where R is a heter-ocyclic ring) and salts thereof. Important members of the sub-class are compounds derived from heterocyclic compounds containing appropriate substituents and which are 5- or 6-membered rings and heterocyclic compounds of this type fused to a 6-rnembered carbocyclic ring, e.g., a benzene ring. Hetero atoms which may be present in the ring include nitrogen, sulphur and oxygen at least one of which must be nitrogen. Usually, the mercapto group will be attached to a carbon atom of the heterocycle and a heterocyclic nitrogen atom will be adjacent to this carbon atom. The heter-ocycle may contain other substituents, e.g., N-alkyl, ketonic oxygen, etc. The sub-class therefore includes derivatives of thiazoline, hydantoin, imidazole, thiazole, oxazole, etc., but it should be understood that it does not include cyclic thioureas.

Examples of heterocyclic nucleophiles which may be used thus include Z-mercaptothiazoline, Z-mercaptohydantoin, l-methyl-2-mercapto-imidazole, Z-mercaptoimidazole, 2-mercapto-benzimidazole, 2-mercapto-benzothiazole, Z-mercapto-benzoxazole, and Z-mercapto-pyridine.

Examples of the group R thus include:

It should be understood that although these heterocyclic compounds are described as thiol (mercapto) or thione compounds they may exist as thione, mercapto zwitterion tautomers and the invention is to be considered as also applying to such tautomeric forms. It will be appreciated that the heterocyclic compounds are described as mercapto or thione compounds for reasons of convention.

(f) Those having the general formula- (in which R and R have the meanings defined above) and their alkali metal salts. Compounds of this type have in general advantageous solubility characteristics in aqueous media and possess improved in vitro activity against gram positive organisms as compared with the parent cephalosporin when the latter is active.

(g) Those having the general formula- (in which R and R have the meanings defined above and R represents the group S O or -HPO and their alkali metal salts.

I 60m H (in which R and R have the meanings defined above) and derivatives thereof substituted in the pyrrole ring, and their alkali metal salts.

The compounds described above will in general have antibacterial activity when at least one acyl group is presout on the amine group in the 7-position and some of such compounds show a superior antibacterial activity against certain organisms than does the parent cephalosporin derivative itself. Compounds which are of particular importance are the thiouronium, substituted thiouronium and azide derivatives. Compounds in which the 7-position is occupied by an unsubstituted amino group are particularly useful as intermediates for the preparation of cephalosporin analogues.

Thus, a compound produced according to the invention which is of particular importance is the thiouronium derivative of benzylcephalosporin which may be represented by the formula:

max. 1 cm.

It showed marked biological activity against Staph. aureus, and B. subtilis. The activity of this derivative against Staph. aureus was found to be superior to that of the parent cephaiosporin C, benzyl cephalosporin and cephalosporin C thiouronium salt. Not only was it found to have significant activity against penicillin sensitive strains of Staph. azn'eus but it was also found to have marked activity against important strains of penicillin resistant Staph. aw'eus. Moreover, it was found to be highly resistant to staphylococcal penicillinase as are of course cephalosporin C and benzylcephalosporin. Another compound which is also important is the thiouronium salt of cephalosporin C itself which was found to be more active against Staph. aureus and B. subtilis than cephalosporin C.

Other important compounds according to the invention having somewhat similar activity to the compound of Formula XI include:

(1) 7-benzylthioacetamidocephalosporin thiouronium salt (2) 7 allylthioacetamidocephalosporin thiouronium (3) 7-/3-benzylthiopropionamidocephalosporin thiouronium salt (5) 7-pentanecarboxamidocephalosporin thiouronium salt (6) 7-n-butylthioacetamidocephalosporin thiouronium salt (7) 7 p-chlorophenylthioacetamidocephalosporin thiouronium salt (8) 7-}3-phenethylthioacetamidocephalosporin thiouronium salt XIX CsH5(CHz)qS.CHz.OO.NH.fiJH-CH (3H2 /NH2 O=CN CCH2SC C N a I CO:

(9) 7-phenylacetamidocephalosporin azide XX CoH5.CH2CO.NH.(I)H-CH CH:

0=G-N C-CHn-Na COzH (10) 7-benzylthioacetamidocephalosporin azide s XXI C6115.CHg.S.CH CO.NH.(EH-?H (I311,

O=CN CCH2N3 1 nium salt XXIII 0611501120 0.NH.CHCH Nrrczr-n (13) 7-phenylacetamidocephalosporin mercaptobenzoxazole derivative XXIV C C-i (III CO2H 011 The compounds obtained according to the invention, may be hydrolysed, particularly those directly obtained from cephalosporin C to produce the corresponding 7- amino cephalosporanic acid derivatives, i.e., having the formula (in which Z is the residue of a strong nucleophile as defined above). These derivatives are important intermediates in the production of acyl derivatives thereof, e.g., phenylacetyl derivatives.

The compounds prepared according to the present invention may be formulated for administration in any convenient Way by analogy with other antibiotic substances.

The compounds may thus be made up into injectable preparations in solution or suspension in a suitable media, 50 e.g., sterile, pyrogen-free water. Alternatively, they may be mixed with solid excipients and then, if desired, compressed into tablets or filled into capsules. They may also be mixed with suitable bases for presentation as suppositories.

Some of the compounds according to the invention are of low solubility and it has been found that the solubility of such compounds can be improved by the use of a physiologically acceptable water soluble amide as solubility promoter.

The water-soluble amide used in accordance with the invention must be physiologically acceptable and thus provoke no undue toxic effects in the proportions used. It is preferred to employ urea or nicotinamide since these substances are physiological substances and in the case of urea the body is provided with an efficient eliminatory mechanism for it whilst nicotinamide is a member of the B complex of vitamins. However, various other watersoluble amides, such as acetamide may also be used which do not provoke any undue toxic effects. It will be appreciated that the administration of a water-soluble amide, particularly by the parenteral route, may give rise to some minor side effects, such as a reduction in blood pressure, but such minor side effects can be tolerated in many cases. In general preference is given to the use of such amides as are solids at 20 C.

Whilst the compositions according to the invention can be formulated for both oral and parenteral administration, they are particularly of value for the formulation of injectable preparations for intramuscular or subcutaneous injections. The preparations can also be formulated as dry mixtures of the antibiotic material together with the amide, which mixtures are adapted to be dissolved or dispersed in pyrogen-free water prior to injection. The preparations may also be formulated for oral administration including, if desired, further excipients, e.g., flavouring and sweetening agents.

Solutions and suspensions of the active substances according to the invention may also contain further solubilising substances, particularly physiologically acceptable water-miscib1e organic solvents for the active material, e. g., alcohol, propylene glycol, dimethylformamide, dimethylacetamide, etc. It will be appreciated that some of these substances are not suitable for parenteral administration whilst being suitable for oral administration and vice versa.

The proportion of amide to antibiotic in the compositions according to the invention varies according to the concentration of the solution which it is desired to achieve. Where it is desired to dissolve the antibiotic in an aqueous solution already containing the amide, it is generally preferable that the solution should be saturated (or nearly saturated) with respect to the amide, as the greater the proportion of amide in the solution, the greater is the solubility of the antibiotic therein. The amount of amide required depends both on the particular amide and antibiotic used but, in general terms, the amide should be present in an amount of at least twice and preferably 20 times the total amount (by weight) of antibiotic present, the solubility of antibiotic generally increasing with increasing proportions of amide.

The compounds according to the invention may be ad ministered in combination with other antibacterial antibiotics especially the penicillins such as penicillin G and/ or the tetracyclines.

In order that the invention may be well understood the following examples are given by way of illustration only. In the examples the following testing and experimental procedure was followed.

PAPER CHROMATOGRAPHY Phosphate bufiered papers.-Anhydrous disodiurn hydrogen phosphate (7.05 g.) in water (2.5 litres; 0.02 M) was adjusted to pH 6 with phosphoric acid; Whatman No. 1 papers (30 x 50 cm.) were dipped into the above solution and dried at 37 overnight.

Sodium acetate bafierezl papers.Hydrated sodium acetate 13.6 g. in water (1 litre; 0.1 M) was adjusted to pH 5 with acetic acid; Whatman No. 1 papers (30 x 30 cm.) were dipped into the above solution and dried.

Paper chromatograms were run on phosphate-buffered paper in (A) butan-l-ol-ethanol-water (4:1:5; by volume) and (B) Propan-l-ol-water (7:3 by volume) also on sodium acetate buffered papers in ethyl acetatesodium acetate solvent system (ethyl acetate saturated with sodium acetate buffer pH 5.0).

Electr0ph0resis.-Electrophoresis was carried out on Whatman 3MM paper at 17 v./cm. (for 2.5-4 hours, unless stated otherwise) in aqueous collidine acetate solution (0.05 M to acetate) pH 7.0 and pyridine acetate solution (0.05 M to acetate) pH 4.0.

Electrophoresis results are expressed as distance travelled by derivative relative to that travelled by Cephalosporin C (or benzylcephalosporin C as the case may be) under the same conditions. A positive value implies migration towards anode, i.e., molecule is negatively charged whereas a negative value indicates migration towards cathode, i.e., positively charged.

Members of the benzyl cephalosporin family were seen as dark spots when the paper was placed before a source of ultra-violet light )\230300 ma) (benzylcephalosporin derivatives do not of course give any coloration with ninhydrin.) They were also detected by means of bioautographs on agar plates inoculated with S. aureus C864 (Oxford H strain), B. subtilis ATCC 6633 or V. cholerae C833 (attenuated laboratory strain).

Members of the cephalosporin C family were seen as dark spots when the paper 'was placed before a source of ultra-violet light (230-300 m and appeared as purple spots when the paper was sprayed with ninhydrin. They were also detected by means of bioautographs on agar plates inoculated with either S. aareas C864 (Oxford H strain) or B sabtilis ATCC 6633.

Example 1 (a) Preparation thiouronium salt of cephalosporin C.- Cephalosporin C (Na salt; 5 g.) together with thiourea (8 g.) were dissolved in distilled water (200 cc.) and allowed to stand in the absence of light at 37 C. for several days. Paper chromatography was used to follow the extent of the reaction. After 4-5 days examination by this technique showed that there was little cephalosporin C remaining. The resulting solution was treated with acetone (1000 cc.) and cooled to 0 C. The oily precipitate which had formed was separated by centrifugation and on further manipulation with acetone gave a pale brown solid (3.7 g.). Under the same conditions a solution of thiourea (concn. 4 g./ 1000 cc.) remained quite clear. The pale brown solid, which contained all the biologically active material, was dissolved in water (50 cc.) and passed through a column (26.0 x 2.5 cm.) of Dowex1 (x 8) in the acetate cycle. The thiouronium salt was eluted rapidly from the column appearing almost at the solvent front. The column was eluted with water and fractions (25 cc.) was collected.

Examination of these fractions by paper chromatography showed only trace amounts of thiourea were present in the solutions of the thiouronium salt, all the unreacted cephalosporin C had been removed. The aqueous solutions containing the thiouronium compound were freeze dried to give a cream solid (2.58 g.). This material was further purified by extraction with absolute methanol (to remove the sodium acetate present) and the thiouronium compound was obtained as a fluffy solid (2.18 g.). Ultra-violet absorption (in H O) A max. v. broad 2590-2640 A. E1 13, 161

The thiouronium salt was obtained as a white microcrystalline solid by careful precipitation from aqueous solution by methanol. Ultra-violet absorption (in H O) A max. v. broad 2590-2640 A. Ef g 189 When subjected to electrophoresis on paper the product behaved as though it had no net charge at pH 7.0 or pH 4.0.

On paper chromatography in butan-l-ol-ethanol-water (B.E.W.), the Rf value (j 0.0-2) of the salt was lower than that of cephalosporin C (0 .03). Similarly, in propan-l-ol-water (P.W.) it had lower Rf (0.11) as compared with cephalosporin C (0.22). Paper chromatography indicated that it yielded both a-amino adipic caid and thiroure-a on acid hydrolysis.

All these properties suggested that the acetoxyl group in cephalosporin C has been replaced by a thiouronium moiety.

(b) Acid hydrolysis of cephalosporin thiouronium salt.Cephalosporin thiouronium salt (50 mg.) was dissolved in hydrochloric acid (2 cc.) at various strengths (a) N/1O (b) 1N (c) 2N (d) 3N (e) 4N (t) 5N and allowed to stand at (i) room temperature cir. 25 C., (ii) 37 C. for several days. Aliquots (10 ,al) were removed at suitable intervals. The reactions were investi gated by paper chromatography and electrophoresis. In all cases the thiouronium compound (Rf 0.11) was converted into a fastermoving material (Rf 0.20) which although very weakly biologically active, against both S.

wureus and B. subtilis was easily detected under U.V. light as a dark zone (as with all cephalosporin derivatives). Spraying the chromatograms with (a) aqueous pyridine followed by (b) acetone solution of phenyl- 12 Continued elution of the column with ammonium acetate buffer (pH 6.8; 0.2 M) displaced the nucleus as a broad peak (tube Nos. 7090). This solution which contained the thiouronium nucleus (shown by paper acetylchloride revealed this faster moving material (Rf chromatography and bioassay after spraying with pyridine 0.20) as a very biologically active material. Similar and phenylacetyl chloride) was phenylacetylated using methods were used to detect this material after eleca larger excess of phenylacetyl chloride, the pH of the trophoresis in which it moved faster towards the cathode solution being maintained at pH 7 by the addition of at pH 4.0 than cephalosporin thiouronium salt. sodium hydrogen carbonate. The resulting solution con- By direct analogy with cephalosporin this material is 10 tained .benzyl cephalosporin thiouronium salt as the only 7-amino cephalosporanic thiouronium salt. From these active material. Direct isolation of the nucleus was not preliminary experiments it appeared that suitable conpossible in this case due to the inability to remove all ditions for the production of 7-amino cephalosporanic the ammonium acetate bufier. thiouronium salt (i.e., thiouronium nucleus) were ob- In Examples 6-19 the following experimental protained when the thiouronium salt was treated with 2-3N cedure was used. hydrochloric acid for 3-4 days. Cephalosporin C Na salt (250 mg. 0.5 mill. mole) (c) Separation of the thiouronium nucleus from acid was dissolved in an aqueous solution (10 cc.) of the hydrolysis of cephalosporin C thiouronium salt.-The nucleophile (5 mill. mole) used. The pH of the solution column and buffers were prepared according to the was from 5.07.5, being adjusted (if necessary) to a value thod of Hi Moore and Stein, J. Biol, Chem. 1952, within this range by the addition of acetic acid. In cer- 195, 669. tain cases where the solubility in water was low, e.g., Cephalosporin C thiouronium salt (500 mg.) was disdiphenyl-thiuorea, the reaction was carried out in 50% solved in aqueous hydrochloric acid (2N; 20 cc.) and (v./v.) dimethylformamide (D.M.F.). The mixture was allowed to stand for three days. The pH of the solution kept at 37 and after diiferent intervals (e.g., 2A, 48 and was then adjusted to pH 4.0 with sodium hydrogen car- 72 hrs.) 5 or 10 1 samples were spotted onto paper bonate. The solution was assayed (biologically) at this for analysis by both electrophoresis and chromatography. point and showed that approx. of the original bio- Bioautographs revealed the new substitution product and logical activity was still present. This solution was remaining cephalosporin C. Similar results were obpassed through a column (2.5 x 30cm.) of Dowex-SO tained after spraying with ninhydrin. (NI-I cycle) and eluted with ammonium formate buffer 30 Electrophoresis results are expressed as distance trav- (pH 4.05; 0.2 M). Fractions of 10 cc. were collected elled by derivative relative to that travelled by parent and assayed according to their ultra-violet absorption. 60 cephalosporin compound under the same conditions. A fractions were collected Nos. 8-28 being the peak fracpositive value implies migration towards the anode, tion. Examination of this material after freeze drying that is the molecule is negatively charged whereas a negashowed that it contained unchanged cephalosporin C tive value indicates migration towards the cathode, thiouronium salt together with other hydrolysis products. i.e., the molecule is positively charged. For convenience No thiouronium nucleus was present in any of these the results of these examples are expressed in tabular fractions. form.

TABLE I Electrophoresis Formula of derivatives Example N ucleophile Reaction Medium B eeph. (in all cases R2=H 0 Value R =HOOC.CH(NHz)(CH2)1.CO)

pH 4.0 pH 7.0

2 Thioureq E20 0.53 0.19 0.26 Formula III R, R R and R =H 3 Phenylthiourea H20 Reaction eflected at 1.39 Formula III C. R =phenyl R5, R6, and R7=H 4 Diphenylthlourea D.M.F./H2O (:30v./v.) 2.43 Formula III R =phenyl R=phenyl R5 and R7=H 5 Ethylenethlourea D.M.F./H:O (70:30v./v.) 0.64 Formula III R4 and RG together=ethylene R5 and R7=H 6 Thioacetamide HzO 1.41 Formula IV R4 and R==H R =methyl 7 Aniline H2O 1.86 -0.l2 +0.69 Formula VI R1=H R2=pl1enyl 8 N-methyl-aniline ELOH/HaO (50:5OV./V.) 2.17 0. 17 +0.05 FormulaVI R =methyl R =phenyl 9 p-Nitroaniline D.M.F./Ha0 (70:30 v./v.) 1. s4 +0.85 +0.72 jgggmfilfivl 10 Sodium p-amino ben E10 0.52 Formula VI R =H R=COONfl TABLE I.-Continued Electrophoresis Formula of derivatives Example Nucleophile Reaction Medium R ceph. (in all cases R =H a Value R =HOOC.CH(NHZ)(CH1):.CO)

pH 4.0 pH 7.0

11 Sodium snlphanilat H20 0. 36 Formula VI R=H R=@SOsNa 12 a-Naphthylamine D.M.F./H2O (70:30 v./v.) 2. 19 Iizogmiiila VI 13 Thiophenol EtOH/HqO (50:50 v./v.). 2. 17 Formula V R =phenyl 14 z-aminoethanethiol H10 0. 42 +0.33 +0. 35 Formula VII R1I=C;H -NH2 15 Sodinmthimnlnhate H10 0. 32 +1. 86 +2. 29 Formula IX R12: S203 Sodium salt 16 Sodium azide ion... H10 1. 14 +1. 02 +1. 04 Formula VIII Sodium salt 17 Sodium phosphate E10 0.66 Formula IX R =HPO4 Sodium hydrogen salt 18 Pyrrole H 1. 73 Formula X 19 p-Nitro-N-methyl aniline D.M.F./H2O 2. 03 Formula VI R10: CH3

R=- N0z 1 Reaction solution also contains additional biologically active compounds.

EXAMPLE 20 (a) Benzylcephalosporin (Na salt; 72 mg.) together with thiourea (128 mg.) were dissolved in distilled water (3 cc.) andallowed to stand in the absence of light at 37 C. for'severaldays. After 3 days, the white precipitate which had formed was removed by centrifugation.

This material was washed with acetone and ether to give a white solid (28.9 mg). A further quantity (approx. 5 mg.) of this material Was obtained from the mother liquors, This solid gave a single biologically active spot on chromatography in the propanolzwater sys-' tem (7:3 by volume). Rf valuev 0.74. R Benzyl ceph. value=0.91.

Examination of the mother liquors by chromatography showed the presence of unchanged benzyl cephalosporin (Na salt) (Rf 0.81), thiourea (Rf 0.50) and small amounts of benzyl cephalosporin thiouronium salt (Rf 0.74). The. benzyl cephalosporin thiouronium salt obtained in this way was very sparingly soluble in water 5 mg./cc.).

Paper electrophoresis showed that it possessed no net charge at pH 7.0. Ultra-violet spectrum (in H 0) 5. max. 2600 A.E{ 189 (b) Phenylacetylation of a mixture obtained according to Example 1(b) in aqueous acetone (50 v./v.) using a large excess of phenylacetyl chloride (i.e., conditions which had been used for the corresponding reaction with cephalosporin C nucleus) gave a solution containing benzylcephalosporin thiouronium salt (identified by its Rf value with an authentic sample) together with other biologically active material (e.g., cephalosporin C thiouronium salt and N-phenylacetyl cephalosporin C 'thiouronium salt).

The overall yield of the conversion of sephalosporin C thiouronium salt to benzyl cephalosporin thiouronium salt is of the order of 4%.

Small-scale production of the memberspf the cephalasporin C family (Examples 21-34) Cephalosporin C Na salt (250 mg; 0.5 mill. mole) was dissolved in an aqueous acetone solution (50 v./v.; 10 cc.) of the nucleophile (1.5 mill. mole, i.e., 3 molar equivalents) employed. The pH of the solution was from 5.0-7.5, being adjusted (if necessary) to a value within this range by the addition of acetic acid or sodium bicarbonate. The mixture was kept at 37 or 50' C. and after difierent intervals (e.g., 24, 48 and 72 hours, etc.) 5 or 10 1. samples were removed for analysis by both electrophoresis and chromatography. Bioautographs revealed the new substitution product and remaining cephalosporin C. Similar results were obtained after spraying with ninhydrin.

Small-scale production of the members of the benzyl.- cephalosporin C family (Examples 354:4)

Due to the overall greater microbiological activity of the benzylcephalosporin C family, and for convenience in investigation solutions more dilute than those above were used, e.g., 7-phenylacetamidocephalosporanic acid (benzylcephalosporin C) as the sodium salt (10 mg.) was dissolved in an aqueous acetone solution (50 v./v.; 2 cc.) of the nucleophile (3 molar equivalents). The reaction was carried out as in the previous example except that aliquots of approx. 1 41. were used for chromatography and electrophoresis.

For convenience the results are expressed in a tabulated form in Tables II and III.

IABLE II.-NUCLEOPHILIC SUBSTITUTION OF CEPHALOSPORIN C R Ceph. value Electrophoresis Example Nucleophile P.W. B.E.W. pH 4.0 pH 7.0

Ethylene thlourea 0.63 O. 27 0. 67 2-rnercaptothiazoline 1. 55 4. 11 2-mercaptohydantoin 1. 64 5. 35 0. 77 0. 20 1-methyI-Z-mereapto-imidazole. 0.75 0. 0. 52 2-mercapto-imidazole 0. 79 0. 16 +0. 39 2-mercaptobenzimidazole 2. O 4. 6 0. 39 +0.22 2-mercaptobenzothiazole. 2. 0 8. 0 +0. 65 O. 54 2-mercaptobenzoxazole. 1. 9 8. 0 +0. 79 +0.50 ZaminothiophenoL 2. 4 10. 0 +0. 80 +0.74 4-aminothiophenol 2. 25 10.0 +0. 04 +0.53 4-nitrothiophenoL 1. 75 8. 0 +0. 93 +0. 69 2,4dinitrothiophenol. 2. 0 4. 0 +0.63 +0.55 Z-aminoethanethiol 0.44 0.2 +0.22 +0.25 3-dimethylaminopropanethiol 0.55 0.4 0. 50 0. 08

l Inseparable from Ceph. 0.

TABLE IIL-NUCLEOPHILIC SUBSTITUTION OF BENZYLCEPHALOSPO RIN C R benzyl Electrophoresis Ceph. 0. value Example Nucleophile B.E.W. Et.Ac. pH 4.0 pH 7.0

Ethylene thiourea 0. 08 0. 17 0.58 2-mercaptothiazoline. 1. 15 3. 06 -0. -1. 20 2-mercaptohydantoin 1. 17 0. 09 0. 20 0. 56 1-methyl-2-mercapto-imidazole 0. 41 0. 14 0. 31 2-mercaptoimidazole 1. 15 0. 0. 22 -0. 19 2-mercaptobenzimidazole 1. 25 1. 9 0. 17 +0. 18 2-mereaptobenzothiazole. 1. 25 3. 5 +0. +0. 29 2mercapt0benzoxazole 1. 25 3. 3 +0. 66 +0. 48 Zaminothiophcnol. 1. 20 3. 35 +0. 27 +0. 54 4-aminothiophcnol 1. 25 3. 4 +0. 0 +0. 0 4-nitrothiophenol. 1. 20 3. 35 +0. 65 +0. 73 Z-aminoethanethio 0.80 0. 05 0. 27 0. 25 3-di.methylaminopropanethiol 0. 75 0. 05 0. 27 0. 25 2-mercapto-4-rnethyl-pyrimidine 1. 1 3. 0 +0. 73 +0. 55 2-arnino-5-mereapto-l,3,4- 1 0. 3 +0.72 +0. 52

thiadiazole. 2-mercaptopy'ridine 1. 15 3. 6 +0.3 Ethyl thiourea 1. 0 0 0 Isopropyl thiourea- 1. 0 0 0 Aniline 1. 23 3.12 0. 12 +0.68 p-Nitroaniline 1. 25 2. 46 +0. 04 +0. 44

I Et.-Ac.-=,ethy1 acetate. 2 Inseparable from benzyl Ceph. 0.

EXAMPLE 55 Preparation of the Z-mercaptobenzoxazole derivative of cephalosporin C cephalosporin C (5 g.) in water (75 cc.) was added with stirring to a solution of Z-mercaptobenzoxazole (5- g.) in acetone (2-15 cc.) and water (60 cc.) and the apparent p-H adjusted from 5 to 6.6 with N-sodium hydrogen carbonate. The mixture (ca 60 v./v. acetone) was kept at 37 for four days when paper chromatography revealed the-presence of only a small amount of cephalosporin C. The reaction mixture was then diluted at 0 to 2 liters with acetone.

Filtration after 3 hours afforded an oil-white solid (4 g.; yield, 80% w./w. cephalosporin C) which was shown by chromatography to contain a small amount (ca 5%) of cephalosporin C. The crude substitution product showed the chromatographic and electrophoretic properties listed in Table II (Example 28). It was very soluble in water.

EXAMPLE 56 Preparation of the Z-mercwptobenz thioazole derivative of benzylcephalosporin C Benzylcephalosporin C sodium salt (435 mg.) in water (4 cc.) was added to a solution of Z-mercaptobenz-othiazole (496 mg.) in acetone (6 cc.) and the apparent pH adjusted to 7.0 with N-sodium hydrogen carbonate. The mixture was kept at for 65 hours when essentially all the starting material had reacted (paper chromatography).

tracted with ethyl acetate at pH 6 in the presence of saturated sodium chloride (5 cc.). An off-white solid (A) separated during the extraction. The residual aqueous solution from this extraction was adjusted to pH 2 and further extracted with ethyl acetate. The pH 6 extract (486 mg.) contained unchanged nucleophile and the desired substitution product. The pH 2' extract (65 mg.) contained traces of unchanged benzyloephalosporin C.

The solid (A) (insoluble in acetone; 136 mg.) was the desired substitution product with the chromatographic and electrophoretic properties listed in Table III (Example 41) and was chromatographically homogeneous.

EXAMPLE 57 Preparation of the 1-methyI-Z-mercapto-imidazole derivative of benzylcep'halosp'orin C Benzylcephalosporin C (Na salt; mg.) was dis solved in water (1 cc.) and treated with a solution of 1- methyl-Z-mercaptoimidazole (85 mg.; 3 molar equivalents) in acetone (1 cc.). The resulting solution was allowed to stand at 37 for several days; the reaction being followed by paper chromatography. After four days the excess of the nucleophile was removed from the reaction mixture by extraction with chloroform (2 x 1 cc.) and the pH of the residual aqueous solution adjusted to circa 2.5 with either phosphoric acid or Amberlite IR. (H+). Extraction with ethyl acetate (3 x 2 cc.) removed the remaining unchanged benzylcephalosporin C.

17 The residual aqueous solution, containing the new substitution product, was adjusted to pH 7 with sodium hydrogen carbonate or Dcacidite G(CO and freeze dried.

Examination by paper chromatography and electro- 1 8 EXAMPLE 59 (a) Preparation of n-heptylcephalosporin.500 mg. of 7ACA were dissolved in 25 mls. 3% NaHCO and 16 mls. of acetone.

5 The solution Was chilled to below 5 C. phoresis showed only one biologically active material, and Inls- InOlq 9 P Y Y 111 i.e., benzylcephalosporin thio-imidazolinium salt, although 0 y acetone added dropwlse Over 30 The varying amounts of inorganic salts were present depend- Sollltlon Was thfin allowed t0 Warm p to room p ing on whether ion-exchange resins were used for the adlure Y 1 110111} acetone Was Removed under vacuo and justment f PH genzylcephalosporin thio imidazolinium 10 the residual solution extracted with 2 x m1. ether The salt was active against both S. aareus and V. cholerae. P Was adlusted to P 3 Prior to beIlZeIle eXtfaCtlOIl x 50 ml.) and then further adjusted to 2.0 before extrac- EXAMPLE 58 tion with ethyl acetate (3 x 50 ml.). Ethyl acetate layers were then bulked, dried over Na SO and reduced in vol- (a) Preparation of phenoxymethyl cephalosporin. 15 ume under vacuum to yield 391 mg. of a crystalline solid. 500 mgm. of 7-aminocephalosporanic acid (7ACA) This was dissolved in a little acetone and 170 mg. (1.1 mol. equiv.) sodium-Z-ethyl hexoate in aqueous acetone l :m. max.== i added. The solution was refrigerated for 1 hr. and the l l t dissolved in 25 mls. of 3% NaHCO;; (5 mol. equiv.) and gg gg g ected by fi tmtlon Washed Wlth dry ace one 16 mls. of acetone. The solution wa chilled to below 29 Yield, mg 5 C. and magnetically stirred while 376 mg. of phenoXyacetyl chloride dissolved in 10 mls. of acetone were added (1) min 220, A dropwlse over 30 mms. The solution was allowed to (2) Chromatography Butano1/EtOH/H2O RF 0J5; warm up to room temperature over 1 hour. The acetone propanol/Hzo RF ethylacetate/bufier R was removed under vacuo and the aqueous layer ex- 29 (3) Electrophoresis pH 7 R :l.0 pH 4.0 tracted with 2 x 25 ml. ether. After ad ustment of the pH Rceph 0:1.O4 to 4.0 with 3N H 50 excess phenoxyacetic acid was extracted With 1 X ITllbenlene- Finally the P 0f the (b) Preparation of n-heptyl cephalosporin thiouronium aqueous layer was adjusted to 2.0 and then extracted with c0rnp0und -15O m of n-hetyl cephalosporin (Na salt) 3 0 1111s ethyl acetate: These eXlractS wer'e bulked, 30 and 70 mg. of thiourea were incubated in 5 ml. H O in drled over 2 4 s) d reduced n um the dark at 37 0. for 3 days. This SOlulZlOn had solidified under vacuum to yield 724 mg. of an oil. This W taken completely at the end of this time and was triturated with p 111 the mmlmum volume f acetone (about 3 n H 0 and the jelly-like material filtered cit, washed with 326 m of Sodlum y heXo-ate mOL q In F H 0 and dried in a vacuum desiccator to give 124 mg. of aqueous acetone was added. The solution was chilled for amorphous powder. 1.5 hr. and the product separated by filtration, washed with dry acetone and dried. 1) U.V.Shoulder 258-265 m;;., but sample not pure.

Yield: 470mg. (2) Chromatography.-Propanol/H O R =0.770; ethyl 40 acetate/butter R =0.1O2. (1) (3) Electr0ph0resz's At pH 4.0 and 7.0, no movement.

1% 1% 126 E1 190 In like manner various other acyl derivatives were pre- (2) Chromatography.Propanol/water (7:3) R 0.73; pared of cephalosporin thiouronium derivative. For conethyl acetate/pH 5 butter R 0.33. venience the results obtained are shown in tabular form (3) Electr0ph0resis.At pH 4.0 and 7.0, Rceph, =1.0. in Tables IV and IVa.

TABLE IV R Benzyl Ceph. Electrophoresis Example Acyl group B.E.W. Et. Ac. H 4.0 pH 7.0

S-phenyl thioacetyl 0.88 0.16

CsH -SCH1C0 61 S-benzyl thioacetyl 0. 92 0.29 0. 11 -0.14

. CsHs-CH:S.CH;CO- 62 Benzoyl 0.80 0.09 63 2,6-dimethoxybenzoy1 1 0.88 0. 17

1 Propanol-water solvent system.

TABLE IVa (b) Preparation of phenoxymethyl cephalosporin thio- 60 UV D t a 3 uranium c0mp0und.-200 mg. of phenoxymethyl cephalo- Example Acyl group sporin (Na salt) and 240 mg. of thiourea were dissolved in XML (mm E1% 2 mls. of H 0 and kept in the dark at 37 C. for 3 days. Onchilling the solution to 0 C. a precipitate was obtained 65 g; g i t l gl gif t 1 ;?2; l 3 which was filtered at 0 C. and washed with a httle 1ce- 7 1 fg f gZ 3 cold water. The solid was dried in a vacuum desiccator 66 fiy op op o v 261-262 200 CsH5CHg.S.CHz.CH2.CO- overnight to yield 67.2 mg. of solid. 67 Phenethylthioacetyl 260 163 oiH oHi.oH .s.0Hi.0o (1) U.V.Shoulder at 258-265 mm, A max. 237 my, 6s n-Butylthioacetyl 260 201 69 Propargylthloacetyl 260 208 7\ min. 230 III/L. ou=o.oH,.s.oHi.oo (2) Chromatography.Propanol/H O 0.665; ethyl ace- 70 p'ohlomphenylthloacetyl 254-256 326 tate buffer 0. Not fully solvent. (3) Electroph0resis.-No movement at pH 4.0 and 7.0. Plateau 19 EXAMPLES 71-79 Preparation of 7-allylthioacetam idocephalosporanic acid sodium salt TABLE VI U.V. Data Example Aeyl groups n-Pentanecarboxy 2G2 242 Benzylthioacetyl 260 216 p-Chlorophenylthioacetyl 255-262 332 The elemental analyses of some compounds prepared in the above examples are given in Table VII:

TABLE VII Found Required Example Formula C H B1 C1 N S C H B1 C1 N S CnHmN4O4SxHzO 44. 3 4. 3 12. 3 21. 8 44. 7 4. 4 12. 3 21. 07

1gI zoN404S3.H2O 45. 5 4- 7 12. 2 20. 9 45. 9 4. 7 11. 9 20. 4 Cr7HzrC1N4O5Sm2HzO 39. 8 4. 09 10. 8 19. 4 40. 1 4. 2 11. 18. 9 C15HzoN404S-2.H3O 44. 1 5. 14. 2 16. 1 44. 8 5. 5 13. 9 15. 9 C 7H 7BLN4O Sa 39. 0 3. 6 10. 9 18. 5 39. 5 3. 4 10. 8 18. 6 C 4HzoN4O5S3.H2O 40. 4 4. 9 13. 2 23. 1 40. O 4. 8 13. 3 22. 9 CHgzN404s2. HzO 45. 8 5. 9 13. 8 15. 1 45. 6 5. 9 14. 2 16. 2 C gHz0N O Sz.HgO 49. 4 5. 5 12. 7 14. 4 49. 3 5. 1 12. 8 14. 6 C 9Hg N O4S3.%HzO 48. D 5. 0 11. 8 20. 7 48. 0 4. 9 11. 8 20. 23 C 4H1sN4O4S3.HgO 39. 9 4. 4 13. 1 22. 9 40. 2 4. 3 13. 4 23. 0 C 7H 7N5O4Sz.%HgO 48. 0 4. 3 16. 4 15. 0 47. 6 4. 2 16. 4 15. 0

in n-butanol (15 ml.) followed by ether (150 ml.). The resultant cream precipitate was filtered off and dried (2.34 g.). crystallisation from aqueous acetone gave the product as a white crystalline solid (1.13 g.), kmax (phosphate bulfer at pH 6) 260 ma (6 9,080). A further crystallisation gave material, 1 (as above) 260 m (6 9,300), [04] +117 (c., 0.87; H O).

The method has also been applied successfully to acylation of 7-aminocephalosporanic acid with cyclopentanoyl chloride, p-bromophenylthioacetyl chloride, n-butanoyl chloride, p-nitrophenylacetyl chloride, n-butanecarboxy chloride, n-pentanecarboxy chloride, trans cinnamoyl chloride and B-phenylpropionyl chloride.

The resulting sodium salts were then reacted in turn with thiourea using the general method described above. The thiouronium salts obtained had the following characteristics:

In a similar manner the azides of n-pentane-carboxamidocephalosporanic acid, benzylthioacetylamidocephalosporanic acid and 7-p-chlorophenylthioacetamidocephalosporanic acid were prepared and gave the following U.V. data.

The activity of various compounds prepared in the above examples against different strains of S. aureus was ascertained by tube dilution assays and compared with related cephalosporin compounds. The results are given in Tables VIII, IX and X.

TABLE VIII Minimum Inhibitory Cohen. /ml.)

Compound Tested S. aureus S. aurcus Oxford 663 Strain Cephalosporin C 62 62 Cephalosporin C thiouronium salt; 8 Product of Example 21 20 Product of Example 26 62 Product of Example 27- 0.6 Product of Example 28 31 Product of Example 29 62 Product of Example 30 31 TABLE IX Minimum Inhibitory Concn. /ml.)

Compound Tested 5 S. aureus 0864 S. aureus Oxford 663 Strain Benzyl cephalosporin 0. 16 0. 16 Benzyleephalosporin thiouronium salt. 0.06 0.06 Product of Example 40 0 08 0.04 Product of Example 41 0.01 Product of Example 42 0 04 0. 04 Product of Example 51 0. 02 Product of Example 52 0.01 Product of Example 53 0. 01 0.01 Product of Example 54 0.02 0. 04

TABLE X S. aureus S. aureus 0864 Oxford 604 Strain Product of Example 60 0.01 0.08 Product of Example 61 0. 02 O. 04 Product of Example 71-. 31 2. 5 Product of Example 72- 0. 01 0.31 Product of Example 0.08 0.31 Product of Example 74. 0.04 0.15 Product of Example 75 0. 01 0.15 Product of Example 76 0. 16 0.62 Product of Example 77 0. 04 0. 16 Product of Example 78. -0. 5 2. 0 Product of Example 79 0.08 0.16 Product of Example 65- 0.05 0. 62 Product of Example 66- 0.01 0. 16 Product of Example 67. 0.08 0.62 Product of Example 68. 0.02 0. 16 Product of Example (l9 0. 04 0.08 Product of Example 70. 0.02 0.08 Product of Example 80 0. 16 0.31 Product of Example 81 0. 04 0.08 Product of Example 82 0. 04 0. 16 Product of Example 20 0.06 0.30 Product of Example 50. 0. 04 0.31 Product of Example 51- Product of Example 42 EXAMPLE 82 Preparation of intramuscular injection (a) Preparation of sterile phenylacetyl cephalosporin thiouronium salt.-Sterile apparatus was used and aseptic conditions were employed throughout.

A 6 percent solution of thiourea in sterile, pyrogenfree water was prepared and also a percent solution of the sodium salt of phenylacetamido ccphalosporanic acid in sterile, pyrogen-free water. Sterile nitrogen was bubbled through each of the solutions to remove any carbon dioxide and the solutions were separately sterilised, by filtration through a 5/3 sintcred glass filter. The sterile solutions were admixed. Air in the container was displaced with nitrogen and the container was sealed. The container was then slowly rotated and maintained at 37 C. for 65 hours. The resulting precipitate was separated by filtration under reduced pressure.

The precipitate was washed with sterile, pyrogen-free water to remove excess thiourea by repeated dispersion in the water until U.V. spectroscopic examination of the precipitate indicated a thiourea content not exceeding 1.5 percent.

The precipitate was dried to constant weight in a desiccator under reduced pressure at ambient temperature. The resulting dried cake was ground to a fine powder in a sterile glass mortar. The resulting sterile powder of phenylacetyl cephalosporin thiouroninm salt (P.A.T.) was then used to prepare intramuscular injections having the following composition:

(b) Preparation of sterile urea-A percent solution of urea in sterile, pynogen-free water was prepared, sterilised by'filtration through a 5/ 3 sintered glass filter and the resulting solution freeze-dried. The freeze-dried cake was reduced to a fine powder by grinding in a sterile glass mortar.

(c) Preparation of injection.-The required quantities of sterile P.A.T. and sterile urea were weighed, uniformly mixed and distributed into sterile 5 ml. vials so that each vial contained 1.1 g. of the mixture. The vials were sealed hermetically to exclude bacteria.

Immediately before the vials are required for use, 1.2 ml. of sterile pyrogen-free water is injected through the vial closure and the vial shaken. In this way 2 mls. of a solution of P.A.T. in urea suitable for intramuscular use is obtained.

22 EXAMPLE 83 Oral Tablet Mg. P.A.T. (fine particle) Maize starch 20 Lactose 74 Polyvinylpyrrolidone 4 Magnesium stear-ate 2 The starch, lactose and P.A.T. is passed through a 60-mesh sieve, dry blended and granulated with a solution of polyvinylpyrrolidone in chloroform. The mass is screened through a 12 mesh sieve and the resulting granules dried at 40 C. to completely remove the chloroform. The dried granules are passed through a 16-mesh screen. The magnesium stearate is mixed with the separated fines and the mixture blended with the granules.

Tablets are manufactured by compression at 200 mg. on punches. The tablets may also be film coated or sugar coated if required.

We claim:

1. A compound selected from the group consisting of compounds of the formula where R is selectedfrom the group consisting of (a) hydrogen (b) triphenyl methyl- (c) lower aralkenoyl (d) propargyl thio acetyl (e) R (CH CO- where R is cycloalkyl, phenyl,

nitrophenyl, chlorophenyl, bromophenyl, lower alkoxy phenyl or lower alkyl phenyl and n is an integer from 1 to 4 (f) R CO- where R contains from 2-7 carbon atoms and is alkyl, alkylthioalkyl, alkoxyalkyl or amino-, carboxy-alkyl Y (g) R CO- where R contains from 2-7 carbon atoms and is alkenyl, alkylthioalkenyl, alkenylthioalkyl, alkoxyalkenyl or alkenyloxyalkyl (h) R X(CH CO where R and n are as defined above and X is oxygen or sulphur (i) R (CH S(CH CH CO where R and n are asglefined above and m. is 0 or an integer from 1 to 4 an (j) R CO-- where R is as defined above and Z is selected from the group consisting of in which R R R and R are selected from the group consisting of hydrogen, alkyl of 2 to 3 carbon atoms, phenyl and ethylene represented by R and R taken together,

in which R and R are selected from the group consisting of amino and nitro,

in which R is selected from the group consisting hydrogen and methyl and R is selected from the group consisting of nitro, carboxyl and --SO H,

new

in which R is alkylene of 2 to carbon atoms,

and Y is selected from the group consisting -COOH, -COOM where M is an alkali metal and in which R is selected from the group consisting of:

(a) hydrogen, (b) triphenyl methyl,

(c) alkanoyl, S-alkylthio alkanoyl and alkoxyalkanoyl of 3 to 8 carbon atoms,

(d) alkenoyl, S-alkenylthio alkanoyl and alkenoxy-alkanoyl of 3 to 8 carbon atoms,

(e) R (CH CO in which n is an integer from 1 to 4 and R is selected from the group consisting of phenyl and nitrophenyl,

(f) phenoxyacetyl,

(g) S-phenylthioacetyl, S chloro-phenylthioacetyl, S- bromo-phenylthioacetyl and S-tertiarybutyl-phenylthioacetyl,

( h) R (CH S(CH CO in which m is an integer from 1 to 4 and n and R have the above given meanmgs,

(i) R CO- in which R is selected from the group consisting of phenyl, cyclopentyl and dimethoxyphenyl,

(j) S-amino-S-carboxylvaleryl (k) propargylthioacetyl and (1) cinnamoyl and Z is selected from the group consisting of:

in which R R R and R are selected from the group consisting of hydrogen, alkyl of 2 to 3 carbon atoms, phenyl and ethylene represented by in which R and R are selected from the group consisting of amino and nitro,

in which R is selected from the group consisting of hydrogen and methyl and R is selected from the group consisting of nitro, carboxyl and SO H,

in which R is alkylene of 2 to 5 carbon atoms, (8) a 2 3 -1 and Y is selected from the group consisting of -COOH, -COOM where M is an alkali metal and when Z is (a) Y is COO 3. A process for the preparation of derivatives of cephalosporin C comprising reacting in a polar medium at a pH of from 5 to 8, a compound selected from the group consisting of compounds of the formula and alkali metal saltsthereof, in which R is lower alkanoyl and R is selected from the group consisting of hydrogen, acyl, and triarylalkyl with at least one molar equivalent of a strong nucleophile selected from the group consisting of:

(a) thioureas of the formula in which R R R and R are selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl and .alkylene represented by R and R taken together,

(b) thioa'mides of the formula in which R R and R have the above given meanings, (c) thiophenols of the formula in which R and R are selected from the group consisting of hydrogen, amino, alkylamino, dialkylamino, nitro, nitroso, carboxyl, cyano and trifluoromethyl,

(d) aromatic amines of the formula in which R and R are selected from the group consisting of hydrogen, amino, alkylamino, dialkylamino, nitro, nitroso, carboxyl, cyano, trifluoromethyl and SO H, and R is selected from the group consisting of hydrogen and alkyl, (e) thiols of the formula in which R is selected from the group consisting of aminoalkyl containing 2 to 5 carbon atoms, 2-thiazolinyl, Z-hydantoinyl, Z-imidazolyl, 2-benzimidazolyl, 2-benzothiazolyl, 2-benzoxazoly1, 2-pyridinyl, S-thiadiazolyl, 2-pyrimidinyl, 2-piperidinyl and amino and alkyl nuclear derivatives of the heterocyclic radicals,

(f) alkali metal salts selected from the group consisting of azides, hydrogen phosphates and thiosulfates and (g) pyrroles and alkyl pyrroles.

4. A process as defined in claim 3 in which said polar medium is water.

5. 7-phenylacetamidocephalosporin thiouronium salt of the formula 6. 7-benzylthioacetamidocephalosporin thiouronium salt of the formula 7. 7-allylthioacetamidocephalosporin thiouronium salt of the formula 9. 7-,6-phenylpropionamidocephalosporin thiouronium salt of the formula 27 10. 7-pentanecarboxamidoceph alosporin thiouroniurn salt of the formula CHs(CH2)4.CO.NH.CH-Cfi CHa NH:

(I) NH1 11. 7-n-butylthioacetamidocephalosporin thiouronium 10 salt of the formula 12. 7-p-chloropheny1thioacetamidocephalosporin thiouronium salt of the formula 13. 7-;8-phenethylthioacetamidocephalosporin thiouronium salt of the formula 14. 7-phenylacetamidocephalosporin azide of the formula @omcounorr-orr EH1 15. 7-benzylthioacetamidocephalosporin azide of the formula 16. 7-phenylacetamidocephalosporin-mercapto pyridine derivative of the formula 17. 7-pheny1acetamidocephalosporin N-ethyl t-hiouronium salt of the formula 18. Phenylacetamidocephalosporin mercaptobenzoxazole derivative of the formula ALEX MAZEL, Primary Examiner.

NICHOLAS S. RIZZO, Examiner.

JAMES W. ADAMS, 111., Assistant Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS OF THE FORMULA 